The overall goal of this project is the development of next-generation functional imaging methods and novel contrast agents that will allow noninvasive measurement of neuroplasticity-related signaling molecules by magnetic resonance imaging (MRI). The work fits into our laboratory's long term agenda of analyzing the function of neural circuitry at a whole-brain level in awake, behaviorally active animals. Our aims will also help establish a broadly significant new generation of functional imaging (fMRI) techniques that combine noninvasiveness and high spatial resolution with readouts specific to neural physiology at the molecular and cellular level, and that may be applied in small animals, primates, and perhaps humans. The proposed research builds on our laboratory's recent introduction of a family of protein-based MRI contrast agents sensitive to an important neuroplasticity-related signaling molecule, the neurotransmitter dopamine (DA). In collaboration with Frances Arnold's group at Caltech, we applied advanced protein engineering methods to create sensors based the heme-binding domain of the bacterial cytochrome P450-BM3 (BM3h). We then used MRI with BM3h-based sensors to detect DA transport in vitro (in PC12 cells) and in vivo (in injected rat brains). Our preliminary results constitute one of the first demonstrations of real-time fMRI with a molecular reporter in vivo, and justify the Specific Aims we propose here. In Specific Aim 1, we will improve on our pilot injection studies, by developing minimally invasive methods for delivery of BM3h-based sensors to large regions of the rodent brain. We will implement two methods for blood brain barrier disruption, in conjunction with intravascular contrast agent delivery. We will also explore gene-directed expression of our protein sensors from cells as a constitutive, potentially targetable delivery strategy. In Specific Aim 2, we will establish robust functional imaging techniques for use with our contrast agents. We will combine MRI pulse sequences for rapid acquisition with flow suppression techniques for removal of hemodynamic artifacts. These methods will improve the sensitivity and confidence with which we can study DA function and its relationship to learning and plasticity in vivo. Sensitivity enhancement will also be achieved by modifying our existing sensors, as part of Specific Aim 3. In Specific Aim 4, we plan to create further BM3h-based sensors for critical extracellular and intracellular signaling molecules related to plasticity in neural systems, exploiting the remarkable versatility of the molecular engineering approach we used to create MRI DA sensors. The work we propose to perform is primarily relevant to two aspects of public health: First, experiments our new methods and reagents will facilitate in animal models will inform our understanding of neuroplasticity processes in humans, and may contribute to the development of treatments for neurological diseases. Second, the reagents we create may eventually be useful as diagnostic imaging tools in clinical practice, once they have proven safe and effective for measuring neural signaling molecules in animals.